Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase

BACKGROUND: Switchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated. RESULTS: A novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used. CONCLUSION: The results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches

Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. “Alamo” with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin

The Switchgrass Genome: Tools and Strategies

Switchgrass ( L.) is a perennial grass species receiving significant focus as a potential bioenergy crop. In the last 5 yr the switchgrass research community has produced a genetic linkage map, an expressed sequence tag (EST) database, a set of single nucleotide polymorphism (SNP) markers that are distributed across the 18 linkage groups, 4x sampling of the AP13 genome in 400-bp reads, and bacterial artificial chromosome (BAC) libraries containing over 200,000 clones. These studies have revealed close collinearity of the switchgrass genome with those of sorghum [ (L.) Moench], rice ( L.), and (L.) P. Beauv. Switchgrass researchers have also developed several microarray technologies for gene expression studies. Switchgrass genomic resources will accelerate the ability of plant breeders to enhance productivity, pest resistance, and nutritional quality. Because switchgrass is a relative newcomer to the genomics world, many secrets of the switchgrass genome have yet to be revealed. To continue to efficiently explore basic and applied topics in switchgrass, it will be critical to capture and exploit the knowledge of plant geneticists and breeders on the next logical steps in the development and utilization of genomic resources for this species. To this end, the community has established a switchgrass genomics executive committee and work group ( [verified 28 Oct. 2011])

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

<p>Abstract</p> <p>Background</p> <p>The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (<it>Panicum virgatum </it>L.) ubiquitin genes (<it>PvUbi1 </it>and <it>PvUbi2</it>) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters) fused to the <it>uidA </it>coding region (<it>GUS</it>) and tested for transient and stable expression in a variety of plant species and tissues.</p> <p>Results</p> <p><it>PvUbi1 </it>consists of 607 bp containing <it>cis</it>-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. <it>PvUbi2 </it>consists of 692 bp containing <it>cis</it>-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. <it>PvUbi1 </it>and <it>PvUbi2 </it>were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV <it>35S, 2x35S, ZmUbi1</it>, and <it>OsAct1 </it>promoters. GUS staining following stable transformation in rice demonstrated that the <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drove expression in all examined tissues. When stably transformed into tobacco (<it>Nicotiana tabacum</it>), the <it>PvUbi2+3 </it>and <it>PvUbi2+9 </it>promoter fusion variants showed expression in vascular and reproductive tissues.</p> <p>Conclusions</p> <p>The <it>PvUbi1 </it>and <it>PvUbi2 </it>promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.</p

Sleep disorders in patients with overweight and acute and exacerbated chronic heart failure

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Medical University - Sofia University Hospital "Tsaritsa Yoanna – ISUL” Background  Often accompanying comorbidity in patients with heart failure and overweight is sleep disorders. Detection and treatment of sleep apnea will be helpful in these patients.  Purpose  To determine the phenotypic characteristics of sleep apnea in these patients. To determine whether there is a link between forms of sleep apnea and type of heart failure.  Methods Hospitalized 46 patients with acute and exacerbate heart failure. Measuring of NT proBNP. Sleep apnea screening with ApneaLink™. Echocardiographic assessment of left ventricular ejection fraction (LVEF) and the E/e‘ ratio. Statistical methods to compare independent samples and correlation analysis for linear dependence.  Results Of the 46 overweight patients with acute and exacerbated chronic heart failure, sleep apnea was diagnosed in 36 patients (78.2%). Of these, 83.3% (n = 30) have оbstructive sleep apnea (OSA) and 16.7% (n = 6) have central sleep apnea (CSA). There was a statistically significant difference in LVEF for the group with CSA (n = 6) vs group with OSA (n = 30) (41.67 ± 13.88 vs. 50.57 ± 8.16, p = 0.038). For diastolic function didn"t reach statistical significance for E/e" ratio (20,33 ± 5,00 vs. 17,06 ± 4,02, p = 0,089). Regarding NTproBNP, there is no significant difference between the groups with OSA and those with CSA (2978.5 ± 2664.1 vs. 2063.36 ± 1877.27 pg/ml, p = 0.316). There is a moderate negative correlation with LVEF and number of central sleep apnea events (r=-0,334, p = 0,047). Conclusions With greater frequency occurs obstructive sleep apnea. Left ventricular systolic function is lower in patients with central sleep apnea. There is a reverse correlation between ejection fraction and number of central apnea. </jats:sec